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Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Stem Cell Reports Report
Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells Ya Meng,1,2 Zhili Ren,1 Faxiang Xu,1 Xiaoxiao Zhou,1 Chengcheng Song,1 Vivien Ya-Fan Wang,2,3 Weiwei Liu,1,4 Ligong Lu,5 James A. Thomson,6,7 and Guokai Chen1,2,* 1Centre
of Reproduction, Development & Aging, Faculty of Health Sciences, University of Macau, Taipa, Macau, China of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 3Cancer Centre, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 4Bioimaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 5Center of Interventional Radiology, Zhuhai Precision Medical Center, Zhuhai People’s Hospital, Jinan University, Zhuhai, Guangdong 519000, China 6Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA 7Department of Cell & Regenerative Biology, University of Wisconsin–Madison, Madison, WI 53706, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stemcr.2018.10.023 2Institute
SUMMARY Nicotinamide, the amide form of vitamin B3, is widely used in disease treatments and stem cell applications. However, nicotinamide’s impact often cannot be attributed to its nutritional functions. In a vitamin screen, we find that nicotinamide promotes cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibits the phosphorylation of myosin light chain, suppresses actomyosin contraction, and leads to improved cell survival after individualization. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and casein kinase 1. We demonstrate that nicotinamide affects human embryonic stem cell pluripotency and differentiation as a selective kinase inhibitor. The findings in this report may help researchers design better strategies to develop nicotinamide-related stem cell applications and disease treatments.
INTRODUCTION Balanced cellular metabolism and signaling regulation are both essential for mammalian cells to survive, proliferate, and function. Nutrients are conventionally thought to act only as enzyme cofactors or energy sources. However, recent advances demonstrate that specific nutrients could also be involved in functions beyond nutritional support, such as epigenetic regulations and kinase cascades (Blaschke et al., 2013; Chen et al., 2013; Gao et al., 2016; Liu et al., 2017; Lu and Thompson, 2012; Sciacovelli et al., 2016; Tzatsos and Kandror, 2006; Yuan et al., 2013). In this report, we explored nicotinamide’s role in stem cell regulation, and showed its regulatory roles as a kinase inhibitor. Nicotinamide is the amide form of niacin, and both of them belong to the vitamin B3 family. They are the precursors of nicotinamide adenine dinucleotide (NAD), which acts as coenzyme in multiple cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted into nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), which is then turned into NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT) (Maiese et al., 2009). The normal plasma concentration of nicotinamide and niacin is around 5 mM (Odum and Wakwe, 2012). Deficiencies in nicotinamide and niacin could lead to decreased NAD+ production and cause pellagra, which affects the skin, digestive system, and CNS (Prakash et al., 2008). Nico-
tinamide, but not niacin, is also an inhibitor of sirtuin and poly(ADP-ribose) polymerase (PARP), which regulate protein deacetylation and DNA repair (Avalos et al., 2005; Jackson et al., 2003; Kuchmerovska et al., 2004; Saldeen and Welsh, 1998). Nicotinamide has been widely used to treat diseases such as diabetes, schizophrenia, Alzheimer’s disease, psoriasis, obesity, and cancer (Aisen et al., 2008; Bagcchi, 2015; Chase et al., 1992; Chen et al., 2015; Gale, 2004; Knip et al., 2000; Siadat et al., 2013; Smythies, 1973). High dosage of nicotinamide is often required in clinical treatment, and the concentration in serum could reach the millimolar range (Dragovic et al., 1995). At the same time, nicotinamide is also used in various cell culture practices. High dosage (more than 10 mM) of nicotinamide promotes cell survival in different cell types, including neural, liver, and heart cells (Ieraci and Herrera, 2006; Lin et al., 2000; Shen et al., 2004; Shi et al., 2012; Tong et al., 2012). Nicotinamide is extensively used in the in vitro culture of organoids, including cell types from colon, liver, pancreas, and fallopian tube (Huch et al., 2013b, 2015; Kessler et al., 2015; Sachs et al., 2018; Sato and Clevers, 2015; Sato et al., 2011; Yin et al., 2016). Nicotinamide also enhances expansion of adult stem cells from pancreas, colon, bone marrow, and umbilical cord (Horwitz et al., 2014; Huch et al., 2013a; Jung et al., 2011; Peled et al., 2012; Sugiyama et al., 2013). In pluripotent stem cells, nicotinamide promotes reprogramming, improves maintenance (Son et al., 2013), and facilitates cell differentiation
Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 j ª 2018 The Authors. 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
to various lineages, including neural, pancreatic, and cardiac lineages (Buchholz et al., 2013; Griffin et al., 2017; Idelson et al., 2009; Nostro et al., 2015; Parsons et al., 2011; Vaca et al., 2008). Despite its numerous applications, the molecular mechanisms of nicotinamide are still unclear in many circumstances. In this study, we set to explore the roles of common vitamins in human pluripotent stem cells (hPSCs), and identified nicotinamide as a regulator of hPSC pluripotency, survival, and differentiation. Nicotinamide promoted hPSC cell survival and differentiation. Further analysis showed that nicotinamide promoted cell survival as a Rho-associated protein kinase (ROCK) inhibitor, while it also inhibited other kinases including casein kinase 1 (CK1) and a few others. Finally, we demonstrated that nicotinamide also initiated differentiation as a kinase inhibitor. Our study revealed the mechanisms underlying nicotinamide’s key functions, and expanded our understanding of its application in cell culture practices.
RESULTS Nicotinamide Promotes hPSC Survival after Individualization through the Regulation of ROCK Pathway hPSCs are vulnerable to cell death after individualization (Chen et al., 2010; Ohgushi et al., 2010). To identify the function of vitamins in stem cell regulation, we tested a set of 12 vitamins at three doses (based on their concentration in DMEM/F12) on cell survival after dissociation in H1 human embryonic stem cells (hESCs) (Figure S1A). Nicotinamide was the only vitamin that promoted hESCs survival after individualization, while high concentrations of retinol and cholecalciferol inhibited cell survival (Figure S1A). The effect of nicotinamide was dose dependent. Nicotinamide promoted survival of individualized cells at 5 and 10 mM, but at 25 mM showed significant toxicity to hESCs (Figure 1A). We then examined cell apoptosis during passage, and found that 10 mM nicotinamide significantly reduced the Annexin V-positive and propidium iodide-negative cells (Figures S1B and S1C). It suggested that nicotinamide suppressed apoptosis, and the observation was consistent with the improved cell survival by nicotinamide. Microscopy images showed that nicotinamide also suppressed the cell blebbing phenotype after dissociation in a dose-dependent manner (Figures 1B and 1C). The beneficial effect was also observed in other pluripotent stem cells (Figures S1D–S1F) as well as on different coating surfaces (Figures S1G and S1H). To understand nicotinamide’s role in cell survival, we tested modulators of a few known nicotinamide targets, 2 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
including sirtuin inhibitors (EX527 and SirReal2) and PARP inhibitor (ABT888). However, neither single inhibitor nor their combination demonstrated the ability to improve cell survival (Figure S1I). It indicates that nicotinamide could function through some other pathways to promote cell survival. It is well known that individualized hESCs were killed through ROCK/actomyosin activation (Chen et al., 2010; Ohgushi et al., 2010). We compared the impact of nicotinamide on cell survival with ROCK inhibitor Y27632. After cell individualization and passaging, nicotinamide improved cell survival with similar efficiency as ROCK inhibitor. However, no additive beneficial effect was observed when they were applied together (Figure 1D), which suggested that nicotinamide and ROCK inhibitor possibly functioned through the same pathway. We then analyzed the impact of nicotinamide on the ROCK pathway. ROCK directly phosphorylates myosin phosphatase-targeting protein (MYPT) at Thr696, and also regulates the phosphorylation of myosin light chain (MLC) directly or indirectly through MYPT (Totsukawa et al., 2000). After dissociation, the phosphorylation of MLC and MYPT increased, and Y27632 and nicotinamide suppressed the phosphorylation of both MLC and MYPT significantly (Figure 1E). This impact of nicotinamide was dose dependent (Figure 1F). Immunostaining results showed that both nicotinamide and Y27632 decreased the colocalization between p-MLC (Ser19) and actin filament after hESC dissociation (Figure 1G). These data indicated that nicotinamide was a modulator of the ROCK pathway. Nicotinamide Is a Direct ROCK Inhibitor Independent of NAD Pathway Nicotinamide is the precursor of NAD+ and NADH, so we tested whether nicotinamide improved cell survival through NAD metabolites. Niacin, NMN, NAD+, and NADH were added to individualized cells, but none of them had significant effect on cell survival (Figure 2A), and these molecules did not block the cell blebbing after individualization (Figure S2A). NAMPT converts nicotinamide into NMN, but NAMPT inhibitors did not alter nicotinamide impact on cell survival (Figures 2B and S2B). Niacin also had no impact on the phosphorylation of MLC and MYPT (Figure 2C). These results suggested that the effects of nicotinamide on cell survival and ROCK pathway regulation were possibly independent of the NAD pathway, and nicotinamide itself might be the direct effector. To study how nicotinamide inhibited ROCK, we tested the protein level of ROCK1 and ROCK2 during dissociation, and found that nicotinamide had no impact (Figure S2C). Then we evaluated the activity of ROCK1 and ROCK2 in vitro with different doses of nicotinamide
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 1. Nicotinamide Promotes hESC Survival through the Inhibition of the ROCK-Actomyosin Axis (A) Dose-dependent effect of nicotinamide on cell survival after dissociation. hESCs (H1 cells unless otherwise stated) were counted 24 hr after individualization. The cell survival index represents the number of surviving cells divided by the input cell number (n = 3). Nam, Nicotinamide. (B) Phase contrast images after individualization. hESCs were dissociated by TrypLE, neutralized by 0.5% BSA, and then treated with the indicated concentration of nicotinamide for 30 min. Scale bar, 20 mm. (C) The percentage of blebbing cells under nicotinamide treatments at different concentrations. The percentage of blebbing cells was normalized by the total cell number (n R 5 images). (D) The comparison of nicotinamide and ROCK inhibitor Y27632 on cell survival after individualization (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM. (E) The phosphorylation of MYPT1 (Thr 696) and MLC (Ser 19) in individualized hESCs under nicotinamide treatment. 10 mM ROCK inhibitor (Y27632) was used as positive control. Top, western blot image. Bottom, quantification of the western blot results (n = 3). (F) Dose-dependent effect of nicotinamide on the phosphorylation of MYPT1 (Thr 696) and MLC (Ser 19). Individualized hESCs were treated with nicotinamide at indicated concentrations for 1 hr. Top, western blot image. Bottom, quantification of the western blot results (n = 3). (G) Confocal images of individualized hESCs treated with 10 mM nicotinamide (Nam) or 10 mM ROCK inhibitor Y27632 (ROCKi). Red, phalloidin 594; green, p-MLC (Ser19). Scale bar, 10 mm. Data are shown as means ± SEM. *p < 0.05 compared with control.
and niacin (Figures 2D and 2E). Surprisingly, the addition of nicotinamide significantly suppressed ROCK1 and ROCK2 activity in a dose-dependent manner, but niacin had almost no effect (Figures 2D and 2E). Computational simulation demonstrated that nicotinamide could potentially interact with key amino acid functional groups in the active site of ROCK2 (Figure S2D). The binding constant assay also confirmed the inhibition of ROCK1 and ROCK2 by nicotinamide (Figures 2F and 2G).
Nicotinamide Regulates More Than the ROCK Pathway ROCK inhibitor Y27632 increases the cloning efficiency of hPSCs (Chen et al., 2010), so we examined the impact of nicotinamide on cloning efficiency. Compared with Y27632, nicotinamide-treated cells showed much smaller improvement in cloning efficiency (Figure 3A), even though both reagents had similar impact on 24-hr cell survival (Figure 1D). High concentrations of nicotinamide decreased the cell growth rate of hESCs (Figure 3B) and Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 3
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 2. Nicotinamide Is a ROCK Inhibitor that Is Independent of the NAD Pathway (A) The comparison of nicotinamide, niacin, NMN, NAD+, and NADH on cell survival after individualization (n = 3). Nam, nicotinamide 10 mM; niacin, 5 mM; NMN, 5 mM; NAD+, 5 mM; NADH, 1 mM. (B) Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 did not block the effect of nicotinamide on cell survival (n = 3). White, FK866 alone; red, FK866 together with 10 mM nicotinamide; blue, FK866 together with 10 mM Y27632. (C) Niacin had no effect on the phosphorylation of MLC and MYPT. Individualized hESCs were treated with niacin at the indicated concentrations for 1 hr (n = 3). (D and E) The impact of nicotinamide on ROCK activity in vitro. ROCK1 (D) and ROCK2 (E) activity was determined by ELISA under nicotinamide (Nam, red curve) or niacin (gray curve) treatment (n = 3 technical replicates). The results were repeated twice. 0.2 mM Y27632 (ROCKi) was used as positive control (black square). (F and G) Binding constant measurements for the interactions of nicotinamide with ROCK1 (F) and ROCK2 (G). The x axis indicates the nicotinamide concentration (mM) in log10 scale (n = 2 technical replicates). Data are shown as means ± SEM. *p < 0.05 compared with control. reduced the mRNA level of NANOG and POU5F1 (Figures 3C and 3D), which indicated that nicotinamide possibly induced hESC differentiation. Among the set of 12 vitamins examined in the differentiation of hESCs, nicotinamide was the only one that affected the pluripotency of hESCs (Figures S3A and S3B). Taken together, this evidence suggests that nicotinamide may have additional functions on pluripotency other than regulating the ROCK pathway. To study the other functions of nicotinamide in hESCs beyond ROCK inhibition, we analyzed the global gene expression profile after 24 hr of nicotinamide and ROCK inhibitor treatment (Table S1). Hierarchical clustering analysis showed that nicotinamide treatment was not clustered with the ROCK inhibitor group (Figure 3E). Compared with control, nicotinamide increased the expression of 371 genes, and decreased the expression of 640 genes after a 24-hr treatment. However, only a small portion of these genes were shared by the cells treated with ROCK inhibitor (Figure S3C). The KEGG analysis showed that the genes downregulated by nicotinamide were enriched in path4 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
ways associated with pluripotency of stem cells, phosphatidylinositol 3-kinase, metabolism, transcription, and cancer (Figure 3F), and the gene expression patterns were different compared with the genes downregulated by the ROCK inhibitor (Figure S3D). The genes upregulated by nicotinamide were also enriched in different pathways from those upregulated by the ROCK inhibitor (Figures S3E and S3F). These data indicated that nicotinamide had multiple functions in hESC regulation. Nicotinamide Affects hESC Differentiation in Multifaceted Manner Because nicotinamide was a direct ROCK inhibitor at high concentration, we hypothesized that nicotinamide might be able to inhibit other kinases. Considering that most nicotinamide effects appeared at 10 mM in cell culture, we measured cellular nicotinamide amounts when 10 mM of nicotinamide was added in the medium. Liquid chromatography-mass spectrometry (LC-MS) results demonstrated that cellular nicotinamide concentration was around
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 3. Nicotinamide Has More Functions Than the ROCK Inhibitor (A) Comparison of ROCK inhibitor Y27632 (ROCKi) and nicotinamide (Nam) on cloning efficiency (n = 3). (B) Dose-dependent effect of nicotinamide on cell growth. hESCs were treated with different doses of nicotinamide, and cell number was counted every day. The fold change was calculated by dividing the cell number by day 0 cell count (n = 3 technical replicates). The results were repeated three times. (C and D) Dose-dependent effect of nicotinamide on pluripotency. NANOG (C) and POU5F1 (D) expression (normalized to control without treatment) were analyzed by qPCR after 3 days of differentiation (n = 3). (E) Hierarchical clustering of samples with indicated treatments in microarray analysis. The phylogenetic relationships of genes are shown on the left, and the cluster relationship of samples is indicated on the top. hESC samples were collected after 24 hr of treatment and compared with untreated control and differentiated mesoderm cells. ROCKi, Y27632 (10 mM for 24 hr). Nam, nicotinamide (10 mM for 24 hr). Mesoderm, hESC-derived mesoderm cells. (F) Bubble plot of enriched KEGG pathways from nicotinamide downregulated genes. Data are shown as means ± SEM. *p < 0.05 compared with control. 1.5 mM after 1 hr of incubation (Figure 4A). Based on the above information, the KINOMEscan assay in a competition-binding method was used to screen the interaction between nicotinamide and the active sites of 97 kinases (Davis et al., 2011; Egan et al., 2015; Fabian et al., 2005; Somoza et al., 2015), and the screening was performed at 1 and
3 mM. We found that multiple kinases were significantly inhibited by nicotinamide (Figure 4B; Table S2). Nicotinamide inhibited 96.7% of kinase-ligand interaction of ROCK2 at 3 mM, which is consistent with the in vitro kinase assay (Figure 2D). It also inhibited 92.3% of kinase-ligand interaction of CK1d (Figure 4B; Table S2). The kinase Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 5
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 4. Nicotinamide Inhibits CK1 and Promotes hESC Differentiation (A) The cellular concentration of nicotinamide determined by LC-MS after 1 hr of treatment. hESCs were cultured in normal E8 medium without additional nicotinamide (Control), or E8 medium with additional 10 mM nicotinamide (Nam) (n = 3). (B) The kinase screening profile for nicotinamide, obtained using the DiscoverRx KINOMEscan service. Nicotinamide was screened at 1 and 3 mM for its ability to inhibit the binding of 97 kinases to substrates in the assay. % Ctrl represents the results of primary screen on binding interactions, and lower numbers indicate stronger hits (see also Table S2). (C–E) Binding constant measurements for the interactions of nicotinamide with CK1d, (C) CK1a (D), and CK13 (E). The x axis indicates the nicotinamide concentration (mM) in log10 scale (n = 2 technical replicates). (legend continued on next page) 6 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
binding constants of nicotinamide with CK1d, CK1a, and CK13 were 352.512, 546.580, and 612.076 mM, respectively (Figures 4C–4E). b-Catenin is the substrate of CK1a, and is specifically phosphorylated at Ser45 (Amit et al., 2002; Liu et al., 2002). We examined b-catenin phosphorylation (Ser45) in hESC, and found that nicotinamide and CK1 inhibitor significantly suppressed Ser45 phosphorylation (Figures 4F and 4G). We also evaluated the impact of nicotinamide on the CK1a activity in vitro using the bioluminescent kinase assay, and the result showed that nicotinamide inhibited CK1a in a dose-dependent manner (Figure 4H). Based on the findings in the kinase screen, we examined whether any of the nicotinamide-inhibited kinases or other targets could affect differentiation in hESCs. hESCs were treated with a set of small molecules that modulated the activities of nicotinamide targets, and allowed to spontaneously differentiate for 3 days. Similar to nicotinamide, CK1 inhibitor D4476 significantly reduced the mRNA level of pluripotency markers (NANOG and POU5F1); in contrast, other inhibitors in ROCK, PARP, and sirtuin pathways did not have a significant impact (Figures 4I and 4J). In embryoid body differentiation, nicotinamide inhibited the expression of meso-endoderm marker genes (MIXL1, TBXT, EOMES, and SOX17), and induced the expression of ectoderm marker genes (PAX6 and NEUROD1). CK1 inhibitor D4476 demonstrated a similar effect (Figure S3G). We also confirmed that both nicotinamide and CK1 inhibitor D4476 blocked meso-endoderm differentiation in BMP4-induced differentiation (Figures S3H–S3J). These results suggest that nicotinamide could lead to hPSC differentiation through the inhibition of CK1. Nicotinamide was reported as an inducer of retinal pigment epithelium (RPE) differentiation (Buchholz et al., 2013), so we explored whether nicotinamide affects RPE differentiation through CK1 inhibition (Figure 4K). Consistent with previous reports, nicotinamide increased the expression of early eye field markers LHX2, PAX6, and RAX on day 6 of RPE differentiation. ROCK inhibitor,
SIRT2 inhibitor, PARP inhibitor, and niacin alone had little effect, while joint treatment with ROCK inhibitor and SIRT1 inhibitor improved the mRNA level of LHX2, PAX6, and RAX, even though the level was much lower than with nicotinamide (Figures S4A–S4C). At the same time, CK1 inhibitor D4476 significantly induced the expression of early eye field markers LHX2, PAX6, and RAX (Figures 4L–4N). The positive impact of nicotinamide, CK1 inhibitor, and CK1/ROCK dual inhibition on RPE differentiation was further confirmed by LHX2 immunostaining (Figure 4O) and flow cytometry analysis (Figure S4D). Similar results were obtained with H9 (Figures S4E–4G) and human induced pluripotent stem cell lines NL1 (Figures S4H–S4J) and NL4 (Figures S4K–S4M). These results indicate that the effect of nicotinamide on RPE differentiation potentially relies on its inhibition on ROCK and CK1 pathways.
DISCUSSION Nicotinamide is widely used in disease treatments and stem cell applications, but many of its effects cannot be explained by its role in nutritional regulation. We demonstrated that nicotinamide regulates stem cell survival and differentiation through the inhibition of specific kinases. Besides its complicated role in metabolism, DNA repair, and epigenetic modification, nicotinamide can modulate various cellular functions through kinase cascades. This is consistent with the diverse applications related to nicotinamide. Nicotinamide has long been used in stem cell culture to improve stem cell performance. Nicotinamide enhanced cell survival and reprogramming, but its function was attributed to its role in the sirtuin pathway and nutritional regulation (Avalos et al., 2005; Son et al., 2013). Our study showed that nicotinamide was a ROCK inhibitor. ROCK inhibitors are known to suppress actomyosin contraction, improve cell survival, and enhance reprogramming
(F) The phosphorylation of b-catenin was decreased by 10 mM nicotinamide or 5 mM CK1 inhibitor D4476 treatment for 6 hr (n = 3) as shown by western blot. (G) Quantification of the western blot results by densitometry (n = 3). (H) The impact of nicotinamide on CK1a activity in vitro. CK1a activity was determined using the bioluminescent kinase assay under nicotinamide (Nam, red curve) treatment (n = 3 technical replicates). The results were repeated three times. 50 mM D4476 (CK1i) was used as positive control (black square). (I and J) Analysis of NANOG (I) and POU5F1 (J) mRNA expression by qPCR after 3 days of spontaneous differentiation. Data are normalized to control (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM; Niacin, 5 mM; SIRT1i, Ex527 10 mM; SIRT2i, SirReal2 500 nM; PARPi, ABT888 50 nM; CK1i, D4476 5 mM. (K) A schematic diagram showing the protocol to differentiate hESCs toward early eye field. (L–N) Nicotinamide and CK1 inhibition promoted the expression of early eye field markers. Expression of LHX2 (L), RAX (M), and PAX6 (N) were analyzed by qPCR (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM; CK1i, D4476 5 mM. (O) Immunostaining of LHX2 (green) on day 12 of RPE differentiation. ROCKi, Y27632 10 mM; Nam, nicotinamide 10 mM; CK1i, D4476 5 mM. Scale bar, 50 mm. Images are representative of three independent experiments. Data are shown as means ± SEM. *p < 0.05 compared with control. Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 7
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
efficiency (Chen et al., 2010; Ohgushi et al., 2010; Watanabe et al., 2007). It is possible that nicotinamide benefits the stem cell culture through its role as a ROCK inhibitor. It is noteworthy that nicotinamide is used in many organoid culture systems, which are also benefited by ROCK inhibition in organoid formation (Miyoshi and Stappenbeck, 2013). The positive effect of nicotinamide on organoid culture may also be related to its role as a ROCK inhibitor. Nicotinamide is used in many different stem cell differentiation platforms, and our data show that this function is likely not based on its inhibition of ROCK. The kinase screen data in this report showed that nicotinamide also inhibited CK1 and other kinases that are associated with pluripotency. In the limited tests, we showed that some of nicotinamide’s impacts on differentiation could potentially be explained by its ability to inhibit CK1 pathways. In RPE differentiation, combination of ROCK and CK1 inhibitors achieved similar effects as nicotinamide alone, which supports our argument that nicotinamide might drive differentiation through CK1 modulation. Unlike nicotinamide, niacin does not inhibit either ROCK or CK1, even though both belong to the vitamin B3 family. It is important to consider their differential impact on cellular functions when people plan to use vitamin B3 for specific treatments. The kinase pathways affected by nicotinamide could provide valuable references for relevant clinical applications. We noticed that nicotinamide was effective in kinase inhibition only at high concentrations, and there is an obvious dose-dependent effect. The high concentration of nicotinamide is often used in disease treatment and cell culture. However, the level of nicotinamide in the serum is much lower. This suggests a dual role of nicotinamide controlled by its cellular concentration. Low-level nicotinamide is sufficient to meet cellular needs as a nutrient, but high concentrations lead to kinase inhibition and subsequently affect survival and differentiation. This partially explains why nicotinamide’s effect on kinase activity was not previously revealed. In summary, this report revealed nicotinamide as a kinase inhibitor regulating stem cell survival and differentiation. These results have practical implications for nicotinamide-related treatments, and provide another angle to further improve its applications.
EXPERIMENTAL PROCEDURES Experimental procedures are also provided in Supplemental Information.
hPSC Culture and Survival Assays The use of hESCs and hiPSCs was approved by the Institutional Review Board at the University of Macau. hESC culture and survival
8 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
assays were carried out as described previously (Chen et al., 2010). See Supplemental Information for more details.
hESC Differentiation to Early RPE Lineage hPSCs were passaged 1:6 on Matrigel (Corning Life Sciences) in E8 medium with 10 mM Y27632 and changed to fresh E8 after cell attachment for 24 hr. Then the differentiation was induced following the methods reported previously with slight modifications (Buchholz et al., 2013). The detailed method is in Supplemental Information.
Statistical Analysis Data are shown as means ± SEM of at least three independent experiments unless otherwise specified, and Student’s t test was used for statistical analysis. p values < 0.05 were considered significant.
ACCESSION NUMBERS The accession number for the microarray data reported in this paper is GEO: GSE121230.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and two tables and can be found with this article online at https://doi.org/10.1016/j.stemcr.2018.10.023.
AUTHOR CONTRIBUTIONS G.C., Y.M., and J.A.T. conceived and designed the study. Y.M., W.L., and G.C. performed the cell survival assays and ROCK-related experiments. F.X. measured intracellular nicotinamide concentration by LC-tandem MS. Y.M. and X.Z. performed hPSC differentiation experiments. Z.R., C.S., and Y.M. prepared samples for microarray and analyzed the data. V.Y.-F.W. contributed to the structure modeling. L.L and Y.M. performed the Annexin V and propidium iodide staining. Y.M., W. L., and G.C. wrote the paper. Most authors contributed to the editing and proofreading of the manuscript.
ACKNOWLEDGMENTS This work was supported by the Science and Technology Development Fund of Macau SAR (FDCT/131/2014/A3 and FDCT/056/ 2015/A2) and University of Macau Multiyear Research Grant (MYRG2015-00228-FHS and MYRG2015-00229-FHS). Received: June 26, 2018 Revised: October 30, 2018 Accepted: October 31, 2018 Published: November 29, 2018
REFERENCES Aisen, P.S., Schneider, L.S., Sano, M., Diaz-Arrastia, R., van Dyck, C.H., Weiner, M.F., Bottiglieri, T., Jin, S., Stokes, K.T., Thomas, R.G., et al. (2008). High-dose B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized controlled trial. JAMA 300, 1774–1783.
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Amit, S., Hatzubai, A., Birman, Y., Andersen, J.S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev. 16, 1066–1076. Avalos, J.L., Bever, K.M., and Wolberger, C. (2005). Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol. Cell 17, 855–868. Bagcchi, S. (2015). Nicotinamide yields impressive results in skin cancer. Lancet Oncol. 16, e591. Blaschke, K., Ebata, K.T., Karimi, M.M., Zepeda-Martinez, J.A., Goyal, P., Mahapatra, S., Tam, A., Laird, D.J., Hirst, M., Rao, A., et al. (2013). Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226. Buchholz, D.E., Pennington, B.O., Croze, R.H., Hinman, C.R., Coffey, P.J., and Clegg, D.O. (2013). Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl. Med. 2, 384–393. Chase, P., Dupre, J., Mahon, J., Ehrlich, R., Gale, E., Kolb, H., Lampeter, E., and Nerup, J. (1992). Nicotinamide and prevention of diabetes. Lancet 339, 1051–1052. Chen, A.C., Martin, A.J., Choy, B., Fernandez-Penas, P., Dalziell, R.A., McKenzie, C.A., Scolyer, R.A., Dhillon, H.M., Vardy, J.L., Kricker, A., et al. (2015). A phase 3 randomized trial of nicotinamide for skin-cancer chemoprevention. N. Engl. J. Med. 373, 1618–1626. Chen, G., Hou, Z., Gulbranson, D.R., and Thomson, J.A. (2010). Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248. Chen, J., Guo, L., Zhang, L., Wu, H., Yang, J., Liu, H., Wang, X., Hu, X., Gu, T., Zhou, Z., et al. (2013). Vitamin C modulates TET1 function during somatic cell reprogramming. Nat. Genet. 45, 1504–1509. Davis, M.I., Hunt, J.P., Herrgard, S., Ciceri, P., Wodicka, L.M., Pallares, G., Hocker, M., Treiber, D.K., and Zarrinkar, P.P. (2011). Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1046–1051. Dragovic, J., Kim, S.H., Brown, S.L., and Kim, J.H. (1995). Nicotinamide pharmacokinetics in patients. Radiother. Oncol. 36, 225–228. Egan, D.F., Chun, M.G., Vamos, M., Zou, H., Rong, J., Miller, C.J., Lou, H.J., Raveendra-panickar, D., Yang, C.C., Sheffler, D.J., et al. (2015). Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Mol. Cell 59, 285–297. Fabian, M.A., Biggs, W.H., Treiber, D.K., Atteridge, C.E., Azimioara, M.D., Benedetti, M.G., Carter, T.A., Ciceri, P., Edeen, P.T., Floyd, M., et al. (2005). A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336. Gale, E.A.M. (2004). European nicotinamide diabetes intervention trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Lancet 363, 925–931. Gao, X., Lin, S.H., Ren, F., Li, J.T., Chen, J.J., Yao, C.B., Yang, H.B., Jiang, S.X., Yan, G.Q., Wang, D., et al. (2016). Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia. Nat. Commun. 7, 11960. Griffin, S.M., Pickard, M.R., Orme, R.P., Hawkins, C.P., Williams, A.C., and Fricker, R.A. (2017). Nicotinamide alone accelerates the
conversion of mouse embryonic stem cells into mature neuronal populations. PLoS One 12, e0183358. Horwitz, M.E., Chao, N.J., Rizzieri, D.A., Long, G.D., Sullivan, K.M., Gasparetto, C., Chute, J.P., Morris, A., McDonald, C., Waters-Pick, B., et al. (2014). Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment. J. Clin. Invest. 124, 3121–3128. Huch, M., Bonfanti, P., Boj, S.F., Sato, T., Loomans, C.J., van de Wetering, M., Sojoodi, M., Li, V.S., Schuijers, J., Gracanin, A., et al. (2013a). Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721. Huch, M., Dorrell, C., Boj, S.F., van Es, J.H., Li, V.S., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M.J., et al. (2013b). In vitro expansion of single Lgr5+ liver stem cells induced by Wntdriven regeneration. Nature 494, 247–250. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M.M., Ellis, E., van Wenum, M., Fuchs, S.A., de Ligt, J., et al. (2015). Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312. Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5, 396–408. Ieraci, A., and Herrera, D.G. (2006). Nicotinamide protects against ethanol-induced apoptotic neurodegeneration in the developing mouse brain. PLoS Med. 3, e101. Jackson, M.D., Schmidt, M.T., Oppenheimer, N.J., and Denu, J.M. (2003). Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases. J. Biol. Chem. 278, 50985–50998. Jung, P., Sato, T., Merlos-Suarez, A., Barriga, F.M., Iglesias, M., Rossell, D., Auer, H., Gallardo, M., Blasco, M.A., Sancho, E., et al. (2011). Isolation and in vitro expansion of human colonic stem cells. Nat. Med. 17, 1225–1227. Kessler, M., Hoffmann, K., Brinkmann, V., Thieck, O., Jackisch, S., Toelle, B., Berger, H., Mollenkopf, H.J., Mangler, M., Sehouli, J., et al. (2015). The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun. 6, 8989. Knip, M., Douek, I.F., Moore, W.P., Gillmor, H.A., McLean, A.E., Bingley, P.J., and Gale, E.A.; European Nicotinamide Diabetes Intervention Trial Group (2000). Safety of high-dose nicotinamide: a review. Diabetologia 43, 1337–1345. Kuchmerovska, T., Shymanskyy, I., Donchenko, G., Kuchmerovskyy, M., Pakirbaieva, L., and Klimenko, A. (2004). Poly(ADP-ribosyl)ation enhancement in brain cell nuclei is associated with diabetic neuropathy. J. Diabetes Complications 18, 198–204. Lin, S.H., Vincent, A., Shaw, T., Maynard, K.I., and Maiese, K. (2000). Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death. J. Cereb. Blood Flow Metab. 20, 1380–1391. Liu, C., Li, Y., Semenov, M., Han, C., Baeg, G.H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002). Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837–847. Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 9
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Liu, P.S., Wang, H., Li, X., Chao, T., Teav, T., Christen, S., Di Conza, G., Cheng, W.C., Chou, C.H., Vavakova, M., et al. (2017). alphaKetoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat. Immunol. 18, 985–994. Lu, C., and Thompson, C.B. (2012). Metabolic regulation of epigenetics. Cell Metab. 16, 9–17. Maiese, K., Chong, Z.Z., Hou, J., and Shang, Y.C. (2009). The vitamin nicotinamide: translating nutrition into clinical care. Molecules 14, 3446–3485. Miyoshi, H., and Stappenbeck, T.S. (2013). In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8, 2471–2482. Nostro, M.C., Sarangi, F., Yang, C., Holland, A., Elefanty, A.G., Stanley, E.G., Greiner, D.L., and Keller, G. (2015). Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports 4, 591–604. Odum, E.P., and Wakwe, V.C. (2012). Plasma concentrations of water-soluble vitamins in metabolic syndrome subjects. Niger. J. Clin. Pract. 15, 442–447. Ohgushi, M., Matsumura, M., Eiraku, M., Murakami, K., Aramaki, T., Nishiyama, A., Muguruma, K., Nakano, T., Suga, H., Ueno, M., et al. (2010). Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225–239. Parsons, X.H., Teng, Y.D., Parsons, J.F., Snyder, E.Y., Smotrich, D.B., and Moore, D.A. (2011). Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction. J. Vis. Exp., e3274. https://doi.org/10.3791/3274. Peled, T., Shoham, H., Aschengrau, D., Yackoubov, D., Frei, G., Rosenheimer, G.N., Lerrer, B., Cohen, H.Y., Nagler, A., Fibach, E., et al. (2012). Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Exp. Hematol. 40, 342–355.e1. Prakash, R., Gandotra, S., Singh, L.K., Das, B., and Lakra, A. (2008). Rapid resolution of delusional parasitosis in pellagra with niacin augmentation therapy. Gen. Hosp. Psychiatry 30, 581–584. Sachs, N., de Ligt, J., Kopper, O., Gogola, E., Bounova, G., Weeber, F., Balgobind, A.V., Wind, K., Gracanin, A., Begthel, H., et al. (2018). A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172, 373–386.e10. Saldeen, J., and Welsh, N. (1998). Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADPribose) polymerase. Mol. Cell. Endocrinol. 139, 99–107. Sato, T., and Clevers, H. (2015). SnapShot: growing organoids from stem cells. Cell 161, 1700.e1. Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., et al. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772. Sciacovelli, M., Goncalves, E., Johnson, T.I., Zecchini, V.R., da Costa, A.S., Gaude, E., Drubbel, A.V., Theobald, S.J., Abbo, S.R.,
10 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
Tran, M.G., et al. (2016). Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition. Nature 537, 544–547. Shen, C.C., Huang, H.M., Ou, H.C., Chen, H.L., Chen, W.C., and Jeng, K.C. (2004). Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation. J. Biomed. Sci. 11, 472–481. Shi, Y., Zhang, L., Jiang, R., Chen, W., Zheng, W., Chen, L., Tang, L., Li, L., Li, L., Tang, W., et al. (2012). Protective effects of nicotinamide against acetaminophen-induced acute liver injury. Int. Immunopharmacol. 14, 530–537. Siadat, A.H., Iraji, F., Khodadadi, M., and Jary, M.K. (2013). Topical nicotinamide in combination with calcipotriol for the treatment of mild to moderate psoriasis: a double-blind, randomized, comparative study. Adv. Biomed. Res. 2, 90. Smythies, J.R. (1973). Letter: nicotinamide treatment of schizophrenia. Lancet 2, 1450–1451. Somoza, J.R., Koditek, D., Villasenor, A.G., Novikov, N., Wong, M.H., Liclican, A., Xing, W., Lagpacan, L., Wang, R., Schultz, B.E., et al. (2015). Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase delta. J. Biol. Chem. 290, 8439–8446. Son, M.J., Son, M.Y., Seol, B., Kim, M.J., Yoo, C.H., Han, M.K., and Cho, Y.S. (2013). Nicotinamide overcomes pluripotency deficits and reprogramming barriers. Stem Cells 31, 1121–1135. Sugiyama, T., Benitez, C.M., Ghodasara, A., Liu, L., McLean, G.W., Lee, J., Blauwkamp, T.A., Nusse, R., Wright, C.V., Gu, G., et al. (2013). Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation. Proc. Natl. Acad. Sci. U S A 110, 12691–12696. Tong, D.L., Zhang, D.X., Xiang, F., Teng, M., Jiang, X.P., Hou, J.M., Zhang, Q., and Huang, Y.S. (2012). Nicotinamide pretreatment protects cardiomyocytes against hypoxia-induced cell death by improving mitochondrial stress. Pharmacology 90, 11–18. Totsukawa, G., Yamakita, Y., Yamashiro, S., Hartshorne, D.J., Sasaki, Y., and Matsumura, F. (2000). Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150, 797–806. Tzatsos, A., and Kandror, K.V. (2006). Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTORmediated insulin receptor substrate 1 phosphorylation. Mol. Cell. Biol. 26, 63–76. Vaca, P., Berna, G., Araujo, R., Carneiro, E.M., Bedoya, F.J., Soria, B., and Martin, F. (2008). Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells. Exp. Cell Res. 314, 969–974. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686. Yin, X., Mead, B.E., Safaee, H., Langer, R., Karp, J.M., and Levy, O. (2016). Engineering stem cell organoids. Cell Stem Cell 18, 25–38. Yuan, H.X., Xiong, Y., and Guan, K.L. (2013). Nutrient sensing, metabolism, and cell growth control. Mol. Cell 49, 379–387.
Stem Cell Reports Report
Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells Ya Meng,1,2 Zhili Ren,1 Faxiang Xu,1 Xiaoxiao Zhou,1 Chengcheng Song,1 Vivien Ya-Fan Wang,2,3 Weiwei Liu,1,4 Ligong Lu,5 James A. Thomson,6,7 and Guokai Chen1,2,* 1Centre
of Reproduction, Development & Aging, Faculty of Health Sciences, University of Macau, Taipa, Macau, China of Translational Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 3Cancer Centre, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 4Bioimaging and Stem Cell Core Facility, Faculty of Health Sciences, University of Macau, Taipa, Macau, China 5Center of Interventional Radiology, Zhuhai Precision Medical Center, Zhuhai People’s Hospital, Jinan University, Zhuhai, Guangdong 519000, China 6Regenerative Biology, Morgridge Institute for Research, Madison, WI 53715, USA 7Department of Cell & Regenerative Biology, University of Wisconsin–Madison, Madison, WI 53706, USA *Correspondence: [email protected] https://doi.org/10.1016/j.stemcr.2018.10.023 2Institute
SUMMARY Nicotinamide, the amide form of vitamin B3, is widely used in disease treatments and stem cell applications. However, nicotinamide’s impact often cannot be attributed to its nutritional functions. In a vitamin screen, we find that nicotinamide promotes cell survival and differentiation in human pluripotent stem cells. Nicotinamide inhibits the phosphorylation of myosin light chain, suppresses actomyosin contraction, and leads to improved cell survival after individualization. Further analysis demonstrates that nicotinamide is an inhibitor of multiple kinases, including ROCK and casein kinase 1. We demonstrate that nicotinamide affects human embryonic stem cell pluripotency and differentiation as a selective kinase inhibitor. The findings in this report may help researchers design better strategies to develop nicotinamide-related stem cell applications and disease treatments.
INTRODUCTION Balanced cellular metabolism and signaling regulation are both essential for mammalian cells to survive, proliferate, and function. Nutrients are conventionally thought to act only as enzyme cofactors or energy sources. However, recent advances demonstrate that specific nutrients could also be involved in functions beyond nutritional support, such as epigenetic regulations and kinase cascades (Blaschke et al., 2013; Chen et al., 2013; Gao et al., 2016; Liu et al., 2017; Lu and Thompson, 2012; Sciacovelli et al., 2016; Tzatsos and Kandror, 2006; Yuan et al., 2013). In this report, we explored nicotinamide’s role in stem cell regulation, and showed its regulatory roles as a kinase inhibitor. Nicotinamide is the amide form of niacin, and both of them belong to the vitamin B3 family. They are the precursors of nicotinamide adenine dinucleotide (NAD), which acts as coenzyme in multiple cellular processes, including energy metabolism and DNA repair. Nicotinamide can be converted into nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT), which is then turned into NAD+ by nicotinamide mononucleotide adenylyltransferase (NMNAT) (Maiese et al., 2009). The normal plasma concentration of nicotinamide and niacin is around 5 mM (Odum and Wakwe, 2012). Deficiencies in nicotinamide and niacin could lead to decreased NAD+ production and cause pellagra, which affects the skin, digestive system, and CNS (Prakash et al., 2008). Nico-
tinamide, but not niacin, is also an inhibitor of sirtuin and poly(ADP-ribose) polymerase (PARP), which regulate protein deacetylation and DNA repair (Avalos et al., 2005; Jackson et al., 2003; Kuchmerovska et al., 2004; Saldeen and Welsh, 1998). Nicotinamide has been widely used to treat diseases such as diabetes, schizophrenia, Alzheimer’s disease, psoriasis, obesity, and cancer (Aisen et al., 2008; Bagcchi, 2015; Chase et al., 1992; Chen et al., 2015; Gale, 2004; Knip et al., 2000; Siadat et al., 2013; Smythies, 1973). High dosage of nicotinamide is often required in clinical treatment, and the concentration in serum could reach the millimolar range (Dragovic et al., 1995). At the same time, nicotinamide is also used in various cell culture practices. High dosage (more than 10 mM) of nicotinamide promotes cell survival in different cell types, including neural, liver, and heart cells (Ieraci and Herrera, 2006; Lin et al., 2000; Shen et al., 2004; Shi et al., 2012; Tong et al., 2012). Nicotinamide is extensively used in the in vitro culture of organoids, including cell types from colon, liver, pancreas, and fallopian tube (Huch et al., 2013b, 2015; Kessler et al., 2015; Sachs et al., 2018; Sato and Clevers, 2015; Sato et al., 2011; Yin et al., 2016). Nicotinamide also enhances expansion of adult stem cells from pancreas, colon, bone marrow, and umbilical cord (Horwitz et al., 2014; Huch et al., 2013a; Jung et al., 2011; Peled et al., 2012; Sugiyama et al., 2013). In pluripotent stem cells, nicotinamide promotes reprogramming, improves maintenance (Son et al., 2013), and facilitates cell differentiation
Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 j ª 2018 The Authors. 1 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
to various lineages, including neural, pancreatic, and cardiac lineages (Buchholz et al., 2013; Griffin et al., 2017; Idelson et al., 2009; Nostro et al., 2015; Parsons et al., 2011; Vaca et al., 2008). Despite its numerous applications, the molecular mechanisms of nicotinamide are still unclear in many circumstances. In this study, we set to explore the roles of common vitamins in human pluripotent stem cells (hPSCs), and identified nicotinamide as a regulator of hPSC pluripotency, survival, and differentiation. Nicotinamide promoted hPSC cell survival and differentiation. Further analysis showed that nicotinamide promoted cell survival as a Rho-associated protein kinase (ROCK) inhibitor, while it also inhibited other kinases including casein kinase 1 (CK1) and a few others. Finally, we demonstrated that nicotinamide also initiated differentiation as a kinase inhibitor. Our study revealed the mechanisms underlying nicotinamide’s key functions, and expanded our understanding of its application in cell culture practices.
RESULTS Nicotinamide Promotes hPSC Survival after Individualization through the Regulation of ROCK Pathway hPSCs are vulnerable to cell death after individualization (Chen et al., 2010; Ohgushi et al., 2010). To identify the function of vitamins in stem cell regulation, we tested a set of 12 vitamins at three doses (based on their concentration in DMEM/F12) on cell survival after dissociation in H1 human embryonic stem cells (hESCs) (Figure S1A). Nicotinamide was the only vitamin that promoted hESCs survival after individualization, while high concentrations of retinol and cholecalciferol inhibited cell survival (Figure S1A). The effect of nicotinamide was dose dependent. Nicotinamide promoted survival of individualized cells at 5 and 10 mM, but at 25 mM showed significant toxicity to hESCs (Figure 1A). We then examined cell apoptosis during passage, and found that 10 mM nicotinamide significantly reduced the Annexin V-positive and propidium iodide-negative cells (Figures S1B and S1C). It suggested that nicotinamide suppressed apoptosis, and the observation was consistent with the improved cell survival by nicotinamide. Microscopy images showed that nicotinamide also suppressed the cell blebbing phenotype after dissociation in a dose-dependent manner (Figures 1B and 1C). The beneficial effect was also observed in other pluripotent stem cells (Figures S1D–S1F) as well as on different coating surfaces (Figures S1G and S1H). To understand nicotinamide’s role in cell survival, we tested modulators of a few known nicotinamide targets, 2 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
including sirtuin inhibitors (EX527 and SirReal2) and PARP inhibitor (ABT888). However, neither single inhibitor nor their combination demonstrated the ability to improve cell survival (Figure S1I). It indicates that nicotinamide could function through some other pathways to promote cell survival. It is well known that individualized hESCs were killed through ROCK/actomyosin activation (Chen et al., 2010; Ohgushi et al., 2010). We compared the impact of nicotinamide on cell survival with ROCK inhibitor Y27632. After cell individualization and passaging, nicotinamide improved cell survival with similar efficiency as ROCK inhibitor. However, no additive beneficial effect was observed when they were applied together (Figure 1D), which suggested that nicotinamide and ROCK inhibitor possibly functioned through the same pathway. We then analyzed the impact of nicotinamide on the ROCK pathway. ROCK directly phosphorylates myosin phosphatase-targeting protein (MYPT) at Thr696, and also regulates the phosphorylation of myosin light chain (MLC) directly or indirectly through MYPT (Totsukawa et al., 2000). After dissociation, the phosphorylation of MLC and MYPT increased, and Y27632 and nicotinamide suppressed the phosphorylation of both MLC and MYPT significantly (Figure 1E). This impact of nicotinamide was dose dependent (Figure 1F). Immunostaining results showed that both nicotinamide and Y27632 decreased the colocalization between p-MLC (Ser19) and actin filament after hESC dissociation (Figure 1G). These data indicated that nicotinamide was a modulator of the ROCK pathway. Nicotinamide Is a Direct ROCK Inhibitor Independent of NAD Pathway Nicotinamide is the precursor of NAD+ and NADH, so we tested whether nicotinamide improved cell survival through NAD metabolites. Niacin, NMN, NAD+, and NADH were added to individualized cells, but none of them had significant effect on cell survival (Figure 2A), and these molecules did not block the cell blebbing after individualization (Figure S2A). NAMPT converts nicotinamide into NMN, but NAMPT inhibitors did not alter nicotinamide impact on cell survival (Figures 2B and S2B). Niacin also had no impact on the phosphorylation of MLC and MYPT (Figure 2C). These results suggested that the effects of nicotinamide on cell survival and ROCK pathway regulation were possibly independent of the NAD pathway, and nicotinamide itself might be the direct effector. To study how nicotinamide inhibited ROCK, we tested the protein level of ROCK1 and ROCK2 during dissociation, and found that nicotinamide had no impact (Figure S2C). Then we evaluated the activity of ROCK1 and ROCK2 in vitro with different doses of nicotinamide
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 1. Nicotinamide Promotes hESC Survival through the Inhibition of the ROCK-Actomyosin Axis (A) Dose-dependent effect of nicotinamide on cell survival after dissociation. hESCs (H1 cells unless otherwise stated) were counted 24 hr after individualization. The cell survival index represents the number of surviving cells divided by the input cell number (n = 3). Nam, Nicotinamide. (B) Phase contrast images after individualization. hESCs were dissociated by TrypLE, neutralized by 0.5% BSA, and then treated with the indicated concentration of nicotinamide for 30 min. Scale bar, 20 mm. (C) The percentage of blebbing cells under nicotinamide treatments at different concentrations. The percentage of blebbing cells was normalized by the total cell number (n R 5 images). (D) The comparison of nicotinamide and ROCK inhibitor Y27632 on cell survival after individualization (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM. (E) The phosphorylation of MYPT1 (Thr 696) and MLC (Ser 19) in individualized hESCs under nicotinamide treatment. 10 mM ROCK inhibitor (Y27632) was used as positive control. Top, western blot image. Bottom, quantification of the western blot results (n = 3). (F) Dose-dependent effect of nicotinamide on the phosphorylation of MYPT1 (Thr 696) and MLC (Ser 19). Individualized hESCs were treated with nicotinamide at indicated concentrations for 1 hr. Top, western blot image. Bottom, quantification of the western blot results (n = 3). (G) Confocal images of individualized hESCs treated with 10 mM nicotinamide (Nam) or 10 mM ROCK inhibitor Y27632 (ROCKi). Red, phalloidin 594; green, p-MLC (Ser19). Scale bar, 10 mm. Data are shown as means ± SEM. *p < 0.05 compared with control.
and niacin (Figures 2D and 2E). Surprisingly, the addition of nicotinamide significantly suppressed ROCK1 and ROCK2 activity in a dose-dependent manner, but niacin had almost no effect (Figures 2D and 2E). Computational simulation demonstrated that nicotinamide could potentially interact with key amino acid functional groups in the active site of ROCK2 (Figure S2D). The binding constant assay also confirmed the inhibition of ROCK1 and ROCK2 by nicotinamide (Figures 2F and 2G).
Nicotinamide Regulates More Than the ROCK Pathway ROCK inhibitor Y27632 increases the cloning efficiency of hPSCs (Chen et al., 2010), so we examined the impact of nicotinamide on cloning efficiency. Compared with Y27632, nicotinamide-treated cells showed much smaller improvement in cloning efficiency (Figure 3A), even though both reagents had similar impact on 24-hr cell survival (Figure 1D). High concentrations of nicotinamide decreased the cell growth rate of hESCs (Figure 3B) and Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 3
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 2. Nicotinamide Is a ROCK Inhibitor that Is Independent of the NAD Pathway (A) The comparison of nicotinamide, niacin, NMN, NAD+, and NADH on cell survival after individualization (n = 3). Nam, nicotinamide 10 mM; niacin, 5 mM; NMN, 5 mM; NAD+, 5 mM; NADH, 1 mM. (B) Nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866 did not block the effect of nicotinamide on cell survival (n = 3). White, FK866 alone; red, FK866 together with 10 mM nicotinamide; blue, FK866 together with 10 mM Y27632. (C) Niacin had no effect on the phosphorylation of MLC and MYPT. Individualized hESCs were treated with niacin at the indicated concentrations for 1 hr (n = 3). (D and E) The impact of nicotinamide on ROCK activity in vitro. ROCK1 (D) and ROCK2 (E) activity was determined by ELISA under nicotinamide (Nam, red curve) or niacin (gray curve) treatment (n = 3 technical replicates). The results were repeated twice. 0.2 mM Y27632 (ROCKi) was used as positive control (black square). (F and G) Binding constant measurements for the interactions of nicotinamide with ROCK1 (F) and ROCK2 (G). The x axis indicates the nicotinamide concentration (mM) in log10 scale (n = 2 technical replicates). Data are shown as means ± SEM. *p < 0.05 compared with control. reduced the mRNA level of NANOG and POU5F1 (Figures 3C and 3D), which indicated that nicotinamide possibly induced hESC differentiation. Among the set of 12 vitamins examined in the differentiation of hESCs, nicotinamide was the only one that affected the pluripotency of hESCs (Figures S3A and S3B). Taken together, this evidence suggests that nicotinamide may have additional functions on pluripotency other than regulating the ROCK pathway. To study the other functions of nicotinamide in hESCs beyond ROCK inhibition, we analyzed the global gene expression profile after 24 hr of nicotinamide and ROCK inhibitor treatment (Table S1). Hierarchical clustering analysis showed that nicotinamide treatment was not clustered with the ROCK inhibitor group (Figure 3E). Compared with control, nicotinamide increased the expression of 371 genes, and decreased the expression of 640 genes after a 24-hr treatment. However, only a small portion of these genes were shared by the cells treated with ROCK inhibitor (Figure S3C). The KEGG analysis showed that the genes downregulated by nicotinamide were enriched in path4 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
ways associated with pluripotency of stem cells, phosphatidylinositol 3-kinase, metabolism, transcription, and cancer (Figure 3F), and the gene expression patterns were different compared with the genes downregulated by the ROCK inhibitor (Figure S3D). The genes upregulated by nicotinamide were also enriched in different pathways from those upregulated by the ROCK inhibitor (Figures S3E and S3F). These data indicated that nicotinamide had multiple functions in hESC regulation. Nicotinamide Affects hESC Differentiation in Multifaceted Manner Because nicotinamide was a direct ROCK inhibitor at high concentration, we hypothesized that nicotinamide might be able to inhibit other kinases. Considering that most nicotinamide effects appeared at 10 mM in cell culture, we measured cellular nicotinamide amounts when 10 mM of nicotinamide was added in the medium. Liquid chromatography-mass spectrometry (LC-MS) results demonstrated that cellular nicotinamide concentration was around
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 3. Nicotinamide Has More Functions Than the ROCK Inhibitor (A) Comparison of ROCK inhibitor Y27632 (ROCKi) and nicotinamide (Nam) on cloning efficiency (n = 3). (B) Dose-dependent effect of nicotinamide on cell growth. hESCs were treated with different doses of nicotinamide, and cell number was counted every day. The fold change was calculated by dividing the cell number by day 0 cell count (n = 3 technical replicates). The results were repeated three times. (C and D) Dose-dependent effect of nicotinamide on pluripotency. NANOG (C) and POU5F1 (D) expression (normalized to control without treatment) were analyzed by qPCR after 3 days of differentiation (n = 3). (E) Hierarchical clustering of samples with indicated treatments in microarray analysis. The phylogenetic relationships of genes are shown on the left, and the cluster relationship of samples is indicated on the top. hESC samples were collected after 24 hr of treatment and compared with untreated control and differentiated mesoderm cells. ROCKi, Y27632 (10 mM for 24 hr). Nam, nicotinamide (10 mM for 24 hr). Mesoderm, hESC-derived mesoderm cells. (F) Bubble plot of enriched KEGG pathways from nicotinamide downregulated genes. Data are shown as means ± SEM. *p < 0.05 compared with control. 1.5 mM after 1 hr of incubation (Figure 4A). Based on the above information, the KINOMEscan assay in a competition-binding method was used to screen the interaction between nicotinamide and the active sites of 97 kinases (Davis et al., 2011; Egan et al., 2015; Fabian et al., 2005; Somoza et al., 2015), and the screening was performed at 1 and
3 mM. We found that multiple kinases were significantly inhibited by nicotinamide (Figure 4B; Table S2). Nicotinamide inhibited 96.7% of kinase-ligand interaction of ROCK2 at 3 mM, which is consistent with the in vitro kinase assay (Figure 2D). It also inhibited 92.3% of kinase-ligand interaction of CK1d (Figure 4B; Table S2). The kinase Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 5
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Figure 4. Nicotinamide Inhibits CK1 and Promotes hESC Differentiation (A) The cellular concentration of nicotinamide determined by LC-MS after 1 hr of treatment. hESCs were cultured in normal E8 medium without additional nicotinamide (Control), or E8 medium with additional 10 mM nicotinamide (Nam) (n = 3). (B) The kinase screening profile for nicotinamide, obtained using the DiscoverRx KINOMEscan service. Nicotinamide was screened at 1 and 3 mM for its ability to inhibit the binding of 97 kinases to substrates in the assay. % Ctrl represents the results of primary screen on binding interactions, and lower numbers indicate stronger hits (see also Table S2). (C–E) Binding constant measurements for the interactions of nicotinamide with CK1d, (C) CK1a (D), and CK13 (E). The x axis indicates the nicotinamide concentration (mM) in log10 scale (n = 2 technical replicates). (legend continued on next page) 6 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
binding constants of nicotinamide with CK1d, CK1a, and CK13 were 352.512, 546.580, and 612.076 mM, respectively (Figures 4C–4E). b-Catenin is the substrate of CK1a, and is specifically phosphorylated at Ser45 (Amit et al., 2002; Liu et al., 2002). We examined b-catenin phosphorylation (Ser45) in hESC, and found that nicotinamide and CK1 inhibitor significantly suppressed Ser45 phosphorylation (Figures 4F and 4G). We also evaluated the impact of nicotinamide on the CK1a activity in vitro using the bioluminescent kinase assay, and the result showed that nicotinamide inhibited CK1a in a dose-dependent manner (Figure 4H). Based on the findings in the kinase screen, we examined whether any of the nicotinamide-inhibited kinases or other targets could affect differentiation in hESCs. hESCs were treated with a set of small molecules that modulated the activities of nicotinamide targets, and allowed to spontaneously differentiate for 3 days. Similar to nicotinamide, CK1 inhibitor D4476 significantly reduced the mRNA level of pluripotency markers (NANOG and POU5F1); in contrast, other inhibitors in ROCK, PARP, and sirtuin pathways did not have a significant impact (Figures 4I and 4J). In embryoid body differentiation, nicotinamide inhibited the expression of meso-endoderm marker genes (MIXL1, TBXT, EOMES, and SOX17), and induced the expression of ectoderm marker genes (PAX6 and NEUROD1). CK1 inhibitor D4476 demonstrated a similar effect (Figure S3G). We also confirmed that both nicotinamide and CK1 inhibitor D4476 blocked meso-endoderm differentiation in BMP4-induced differentiation (Figures S3H–S3J). These results suggest that nicotinamide could lead to hPSC differentiation through the inhibition of CK1. Nicotinamide was reported as an inducer of retinal pigment epithelium (RPE) differentiation (Buchholz et al., 2013), so we explored whether nicotinamide affects RPE differentiation through CK1 inhibition (Figure 4K). Consistent with previous reports, nicotinamide increased the expression of early eye field markers LHX2, PAX6, and RAX on day 6 of RPE differentiation. ROCK inhibitor,
SIRT2 inhibitor, PARP inhibitor, and niacin alone had little effect, while joint treatment with ROCK inhibitor and SIRT1 inhibitor improved the mRNA level of LHX2, PAX6, and RAX, even though the level was much lower than with nicotinamide (Figures S4A–S4C). At the same time, CK1 inhibitor D4476 significantly induced the expression of early eye field markers LHX2, PAX6, and RAX (Figures 4L–4N). The positive impact of nicotinamide, CK1 inhibitor, and CK1/ROCK dual inhibition on RPE differentiation was further confirmed by LHX2 immunostaining (Figure 4O) and flow cytometry analysis (Figure S4D). Similar results were obtained with H9 (Figures S4E–4G) and human induced pluripotent stem cell lines NL1 (Figures S4H–S4J) and NL4 (Figures S4K–S4M). These results indicate that the effect of nicotinamide on RPE differentiation potentially relies on its inhibition on ROCK and CK1 pathways.
DISCUSSION Nicotinamide is widely used in disease treatments and stem cell applications, but many of its effects cannot be explained by its role in nutritional regulation. We demonstrated that nicotinamide regulates stem cell survival and differentiation through the inhibition of specific kinases. Besides its complicated role in metabolism, DNA repair, and epigenetic modification, nicotinamide can modulate various cellular functions through kinase cascades. This is consistent with the diverse applications related to nicotinamide. Nicotinamide has long been used in stem cell culture to improve stem cell performance. Nicotinamide enhanced cell survival and reprogramming, but its function was attributed to its role in the sirtuin pathway and nutritional regulation (Avalos et al., 2005; Son et al., 2013). Our study showed that nicotinamide was a ROCK inhibitor. ROCK inhibitors are known to suppress actomyosin contraction, improve cell survival, and enhance reprogramming
(F) The phosphorylation of b-catenin was decreased by 10 mM nicotinamide or 5 mM CK1 inhibitor D4476 treatment for 6 hr (n = 3) as shown by western blot. (G) Quantification of the western blot results by densitometry (n = 3). (H) The impact of nicotinamide on CK1a activity in vitro. CK1a activity was determined using the bioluminescent kinase assay under nicotinamide (Nam, red curve) treatment (n = 3 technical replicates). The results were repeated three times. 50 mM D4476 (CK1i) was used as positive control (black square). (I and J) Analysis of NANOG (I) and POU5F1 (J) mRNA expression by qPCR after 3 days of spontaneous differentiation. Data are normalized to control (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM; Niacin, 5 mM; SIRT1i, Ex527 10 mM; SIRT2i, SirReal2 500 nM; PARPi, ABT888 50 nM; CK1i, D4476 5 mM. (K) A schematic diagram showing the protocol to differentiate hESCs toward early eye field. (L–N) Nicotinamide and CK1 inhibition promoted the expression of early eye field markers. Expression of LHX2 (L), RAX (M), and PAX6 (N) were analyzed by qPCR (n = 3). Nam, nicotinamide 10 mM; ROCKi, Y27632 10 mM; CK1i, D4476 5 mM. (O) Immunostaining of LHX2 (green) on day 12 of RPE differentiation. ROCKi, Y27632 10 mM; Nam, nicotinamide 10 mM; CK1i, D4476 5 mM. Scale bar, 50 mm. Images are representative of three independent experiments. Data are shown as means ± SEM. *p < 0.05 compared with control. Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 7
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
efficiency (Chen et al., 2010; Ohgushi et al., 2010; Watanabe et al., 2007). It is possible that nicotinamide benefits the stem cell culture through its role as a ROCK inhibitor. It is noteworthy that nicotinamide is used in many organoid culture systems, which are also benefited by ROCK inhibition in organoid formation (Miyoshi and Stappenbeck, 2013). The positive effect of nicotinamide on organoid culture may also be related to its role as a ROCK inhibitor. Nicotinamide is used in many different stem cell differentiation platforms, and our data show that this function is likely not based on its inhibition of ROCK. The kinase screen data in this report showed that nicotinamide also inhibited CK1 and other kinases that are associated with pluripotency. In the limited tests, we showed that some of nicotinamide’s impacts on differentiation could potentially be explained by its ability to inhibit CK1 pathways. In RPE differentiation, combination of ROCK and CK1 inhibitors achieved similar effects as nicotinamide alone, which supports our argument that nicotinamide might drive differentiation through CK1 modulation. Unlike nicotinamide, niacin does not inhibit either ROCK or CK1, even though both belong to the vitamin B3 family. It is important to consider their differential impact on cellular functions when people plan to use vitamin B3 for specific treatments. The kinase pathways affected by nicotinamide could provide valuable references for relevant clinical applications. We noticed that nicotinamide was effective in kinase inhibition only at high concentrations, and there is an obvious dose-dependent effect. The high concentration of nicotinamide is often used in disease treatment and cell culture. However, the level of nicotinamide in the serum is much lower. This suggests a dual role of nicotinamide controlled by its cellular concentration. Low-level nicotinamide is sufficient to meet cellular needs as a nutrient, but high concentrations lead to kinase inhibition and subsequently affect survival and differentiation. This partially explains why nicotinamide’s effect on kinase activity was not previously revealed. In summary, this report revealed nicotinamide as a kinase inhibitor regulating stem cell survival and differentiation. These results have practical implications for nicotinamide-related treatments, and provide another angle to further improve its applications.
EXPERIMENTAL PROCEDURES Experimental procedures are also provided in Supplemental Information.
hPSC Culture and Survival Assays The use of hESCs and hiPSCs was approved by the Institutional Review Board at the University of Macau. hESC culture and survival
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assays were carried out as described previously (Chen et al., 2010). See Supplemental Information for more details.
hESC Differentiation to Early RPE Lineage hPSCs were passaged 1:6 on Matrigel (Corning Life Sciences) in E8 medium with 10 mM Y27632 and changed to fresh E8 after cell attachment for 24 hr. Then the differentiation was induced following the methods reported previously with slight modifications (Buchholz et al., 2013). The detailed method is in Supplemental Information.
Statistical Analysis Data are shown as means ± SEM of at least three independent experiments unless otherwise specified, and Student’s t test was used for statistical analysis. p values < 0.05 were considered significant.
ACCESSION NUMBERS The accession number for the microarray data reported in this paper is GEO: GSE121230.
SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, four figures, and two tables and can be found with this article online at https://doi.org/10.1016/j.stemcr.2018.10.023.
AUTHOR CONTRIBUTIONS G.C., Y.M., and J.A.T. conceived and designed the study. Y.M., W.L., and G.C. performed the cell survival assays and ROCK-related experiments. F.X. measured intracellular nicotinamide concentration by LC-tandem MS. Y.M. and X.Z. performed hPSC differentiation experiments. Z.R., C.S., and Y.M. prepared samples for microarray and analyzed the data. V.Y.-F.W. contributed to the structure modeling. L.L and Y.M. performed the Annexin V and propidium iodide staining. Y.M., W. L., and G.C. wrote the paper. Most authors contributed to the editing and proofreading of the manuscript.
ACKNOWLEDGMENTS This work was supported by the Science and Technology Development Fund of Macau SAR (FDCT/131/2014/A3 and FDCT/056/ 2015/A2) and University of Macau Multiyear Research Grant (MYRG2015-00228-FHS and MYRG2015-00229-FHS). Received: June 26, 2018 Revised: October 30, 2018 Accepted: October 31, 2018 Published: November 29, 2018
REFERENCES Aisen, P.S., Schneider, L.S., Sano, M., Diaz-Arrastia, R., van Dyck, C.H., Weiner, M.F., Bottiglieri, T., Jin, S., Stokes, K.T., Thomas, R.G., et al. (2008). High-dose B vitamin supplementation and cognitive decline in Alzheimer disease: a randomized controlled trial. JAMA 300, 1774–1783.
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Amit, S., Hatzubai, A., Birman, Y., Andersen, J.S., Ben-Shushan, E., Mann, M., Ben-Neriah, Y., and Alkalay, I. (2002). Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev. 16, 1066–1076. Avalos, J.L., Bever, K.M., and Wolberger, C. (2005). Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol. Cell 17, 855–868. Bagcchi, S. (2015). Nicotinamide yields impressive results in skin cancer. Lancet Oncol. 16, e591. Blaschke, K., Ebata, K.T., Karimi, M.M., Zepeda-Martinez, J.A., Goyal, P., Mahapatra, S., Tam, A., Laird, D.J., Hirst, M., Rao, A., et al. (2013). Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. Nature 500, 222–226. Buchholz, D.E., Pennington, B.O., Croze, R.H., Hinman, C.R., Coffey, P.J., and Clegg, D.O. (2013). Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl. Med. 2, 384–393. Chase, P., Dupre, J., Mahon, J., Ehrlich, R., Gale, E., Kolb, H., Lampeter, E., and Nerup, J. (1992). Nicotinamide and prevention of diabetes. Lancet 339, 1051–1052. Chen, A.C., Martin, A.J., Choy, B., Fernandez-Penas, P., Dalziell, R.A., McKenzie, C.A., Scolyer, R.A., Dhillon, H.M., Vardy, J.L., Kricker, A., et al. (2015). A phase 3 randomized trial of nicotinamide for skin-cancer chemoprevention. N. Engl. J. Med. 373, 1618–1626. Chen, G., Hou, Z., Gulbranson, D.R., and Thomson, J.A. (2010). Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240–248. Chen, J., Guo, L., Zhang, L., Wu, H., Yang, J., Liu, H., Wang, X., Hu, X., Gu, T., Zhou, Z., et al. (2013). Vitamin C modulates TET1 function during somatic cell reprogramming. Nat. Genet. 45, 1504–1509. Davis, M.I., Hunt, J.P., Herrgard, S., Ciceri, P., Wodicka, L.M., Pallares, G., Hocker, M., Treiber, D.K., and Zarrinkar, P.P. (2011). Comprehensive analysis of kinase inhibitor selectivity. Nat. Biotechnol. 29, 1046–1051. Dragovic, J., Kim, S.H., Brown, S.L., and Kim, J.H. (1995). Nicotinamide pharmacokinetics in patients. Radiother. Oncol. 36, 225–228. Egan, D.F., Chun, M.G., Vamos, M., Zou, H., Rong, J., Miller, C.J., Lou, H.J., Raveendra-panickar, D., Yang, C.C., Sheffler, D.J., et al. (2015). Small molecule inhibition of the autophagy kinase ULK1 and identification of ULK1 substrates. Mol. Cell 59, 285–297. Fabian, M.A., Biggs, W.H., Treiber, D.K., Atteridge, C.E., Azimioara, M.D., Benedetti, M.G., Carter, T.A., Ciceri, P., Edeen, P.T., Floyd, M., et al. (2005). A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 23, 329–336. Gale, E.A.M. (2004). European nicotinamide diabetes intervention trial (ENDIT): a randomised controlled trial of intervention before the onset of type 1 diabetes. Lancet 363, 925–931. Gao, X., Lin, S.H., Ren, F., Li, J.T., Chen, J.J., Yao, C.B., Yang, H.B., Jiang, S.X., Yan, G.Q., Wang, D., et al. (2016). Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia. Nat. Commun. 7, 11960. Griffin, S.M., Pickard, M.R., Orme, R.P., Hawkins, C.P., Williams, A.C., and Fricker, R.A. (2017). Nicotinamide alone accelerates the
conversion of mouse embryonic stem cells into mature neuronal populations. PLoS One 12, e0183358. Horwitz, M.E., Chao, N.J., Rizzieri, D.A., Long, G.D., Sullivan, K.M., Gasparetto, C., Chute, J.P., Morris, A., McDonald, C., Waters-Pick, B., et al. (2014). Umbilical cord blood expansion with nicotinamide provides long-term multilineage engraftment. J. Clin. Invest. 124, 3121–3128. Huch, M., Bonfanti, P., Boj, S.F., Sato, T., Loomans, C.J., van de Wetering, M., Sojoodi, M., Li, V.S., Schuijers, J., Gracanin, A., et al. (2013a). Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis. EMBO J. 32, 2708–2721. Huch, M., Dorrell, C., Boj, S.F., van Es, J.H., Li, V.S., van de Wetering, M., Sato, T., Hamer, K., Sasaki, N., Finegold, M.J., et al. (2013b). In vitro expansion of single Lgr5+ liver stem cells induced by Wntdriven regeneration. Nature 494, 247–250. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M.M., Ellis, E., van Wenum, M., Fuchs, S.A., de Ligt, J., et al. (2015). Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312. Idelson, M., Alper, R., Obolensky, A., Ben-Shushan, E., Hemo, I., Yachimovich-Cohen, N., Khaner, H., Smith, Y., Wiser, O., Gropp, M., et al. (2009). Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5, 396–408. Ieraci, A., and Herrera, D.G. (2006). Nicotinamide protects against ethanol-induced apoptotic neurodegeneration in the developing mouse brain. PLoS Med. 3, e101. Jackson, M.D., Schmidt, M.T., Oppenheimer, N.J., and Denu, J.M. (2003). Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases. J. Biol. Chem. 278, 50985–50998. Jung, P., Sato, T., Merlos-Suarez, A., Barriga, F.M., Iglesias, M., Rossell, D., Auer, H., Gallardo, M., Blasco, M.A., Sancho, E., et al. (2011). Isolation and in vitro expansion of human colonic stem cells. Nat. Med. 17, 1225–1227. Kessler, M., Hoffmann, K., Brinkmann, V., Thieck, O., Jackisch, S., Toelle, B., Berger, H., Mollenkopf, H.J., Mangler, M., Sehouli, J., et al. (2015). The Notch and Wnt pathways regulate stemness and differentiation in human fallopian tube organoids. Nat. Commun. 6, 8989. Knip, M., Douek, I.F., Moore, W.P., Gillmor, H.A., McLean, A.E., Bingley, P.J., and Gale, E.A.; European Nicotinamide Diabetes Intervention Trial Group (2000). Safety of high-dose nicotinamide: a review. Diabetologia 43, 1337–1345. Kuchmerovska, T., Shymanskyy, I., Donchenko, G., Kuchmerovskyy, M., Pakirbaieva, L., and Klimenko, A. (2004). Poly(ADP-ribosyl)ation enhancement in brain cell nuclei is associated with diabetic neuropathy. J. Diabetes Complications 18, 198–204. Lin, S.H., Vincent, A., Shaw, T., Maynard, K.I., and Maiese, K. (2000). Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death. J. Cereb. Blood Flow Metab. 20, 1380–1391. Liu, C., Li, Y., Semenov, M., Han, C., Baeg, G.H., Tan, Y., Zhang, Z., Lin, X., and He, X. (2002). Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837–847. Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018 9
Please cite this article in press as: Meng et al., Nicotinamide Promotes Cell Survival and Differentiation as Kinase Inhibitor in Human Pluripotent Stem Cells, Stem Cell Reports (2018), https://doi.org/10.1016/j.stemcr.2018.10.023
Liu, P.S., Wang, H., Li, X., Chao, T., Teav, T., Christen, S., Di Conza, G., Cheng, W.C., Chou, C.H., Vavakova, M., et al. (2017). alphaKetoglutarate orchestrates macrophage activation through metabolic and epigenetic reprogramming. Nat. Immunol. 18, 985–994. Lu, C., and Thompson, C.B. (2012). Metabolic regulation of epigenetics. Cell Metab. 16, 9–17. Maiese, K., Chong, Z.Z., Hou, J., and Shang, Y.C. (2009). The vitamin nicotinamide: translating nutrition into clinical care. Molecules 14, 3446–3485. Miyoshi, H., and Stappenbeck, T.S. (2013). In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat. Protoc. 8, 2471–2482. Nostro, M.C., Sarangi, F., Yang, C., Holland, A., Elefanty, A.G., Stanley, E.G., Greiner, D.L., and Keller, G. (2015). Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines. Stem Cell Reports 4, 591–604. Odum, E.P., and Wakwe, V.C. (2012). Plasma concentrations of water-soluble vitamins in metabolic syndrome subjects. Niger. J. Clin. Pract. 15, 442–447. Ohgushi, M., Matsumura, M., Eiraku, M., Murakami, K., Aramaki, T., Nishiyama, A., Muguruma, K., Nakano, T., Suga, H., Ueno, M., et al. (2010). Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225–239. Parsons, X.H., Teng, Y.D., Parsons, J.F., Snyder, E.Y., Smotrich, D.B., and Moore, D.A. (2011). Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction. J. Vis. Exp., e3274. https://doi.org/10.3791/3274. Peled, T., Shoham, H., Aschengrau, D., Yackoubov, D., Frei, G., Rosenheimer, G.N., Lerrer, B., Cohen, H.Y., Nagler, A., Fibach, E., et al. (2012). Nicotinamide, a SIRT1 inhibitor, inhibits differentiation and facilitates expansion of hematopoietic progenitor cells with enhanced bone marrow homing and engraftment. Exp. Hematol. 40, 342–355.e1. Prakash, R., Gandotra, S., Singh, L.K., Das, B., and Lakra, A. (2008). Rapid resolution of delusional parasitosis in pellagra with niacin augmentation therapy. Gen. Hosp. Psychiatry 30, 581–584. Sachs, N., de Ligt, J., Kopper, O., Gogola, E., Bounova, G., Weeber, F., Balgobind, A.V., Wind, K., Gracanin, A., Begthel, H., et al. (2018). A living biobank of breast cancer organoids captures disease heterogeneity. Cell 172, 373–386.e10. Saldeen, J., and Welsh, N. (1998). Nicotinamide-induced apoptosis in insulin producing cells is associated with cleavage of poly(ADPribose) polymerase. Mol. Cell. Endocrinol. 139, 99–107. Sato, T., and Clevers, H. (2015). SnapShot: growing organoids from stem cells. Cell 161, 1700.e1. Sato, T., Stange, D.E., Ferrante, M., Vries, R.G., Van Es, J.H., Van den Brink, S., Van Houdt, W.J., Pronk, A., Van Gorp, J., Siersema, P.D., et al. (2011). Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772. Sciacovelli, M., Goncalves, E., Johnson, T.I., Zecchini, V.R., da Costa, A.S., Gaude, E., Drubbel, A.V., Theobald, S.J., Abbo, S.R.,
10 Stem Cell Reports j Vol. 11 j 1–10 j December 11, 2018
Tran, M.G., et al. (2016). Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition. Nature 537, 544–547. Shen, C.C., Huang, H.M., Ou, H.C., Chen, H.L., Chen, W.C., and Jeng, K.C. (2004). Protective effect of nicotinamide on neuronal cells under oxygen and glucose deprivation and hypoxia/reoxygenation. J. Biomed. Sci. 11, 472–481. Shi, Y., Zhang, L., Jiang, R., Chen, W., Zheng, W., Chen, L., Tang, L., Li, L., Li, L., Tang, W., et al. (2012). Protective effects of nicotinamide against acetaminophen-induced acute liver injury. Int. Immunopharmacol. 14, 530–537. Siadat, A.H., Iraji, F., Khodadadi, M., and Jary, M.K. (2013). Topical nicotinamide in combination with calcipotriol for the treatment of mild to moderate psoriasis: a double-blind, randomized, comparative study. Adv. Biomed. Res. 2, 90. Smythies, J.R. (1973). Letter: nicotinamide treatment of schizophrenia. Lancet 2, 1450–1451. Somoza, J.R., Koditek, D., Villasenor, A.G., Novikov, N., Wong, M.H., Liclican, A., Xing, W., Lagpacan, L., Wang, R., Schultz, B.E., et al. (2015). Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase delta. J. Biol. Chem. 290, 8439–8446. Son, M.J., Son, M.Y., Seol, B., Kim, M.J., Yoo, C.H., Han, M.K., and Cho, Y.S. (2013). Nicotinamide overcomes pluripotency deficits and reprogramming barriers. Stem Cells 31, 1121–1135. Sugiyama, T., Benitez, C.M., Ghodasara, A., Liu, L., McLean, G.W., Lee, J., Blauwkamp, T.A., Nusse, R., Wright, C.V., Gu, G., et al. (2013). Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation. Proc. Natl. Acad. Sci. U S A 110, 12691–12696. Tong, D.L., Zhang, D.X., Xiang, F., Teng, M., Jiang, X.P., Hou, J.M., Zhang, Q., and Huang, Y.S. (2012). Nicotinamide pretreatment protects cardiomyocytes against hypoxia-induced cell death by improving mitochondrial stress. Pharmacology 90, 11–18. Totsukawa, G., Yamakita, Y., Yamashiro, S., Hartshorne, D.J., Sasaki, Y., and Matsumura, F. (2000). Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150, 797–806. Tzatsos, A., and Kandror, K.V. (2006). Nutrients suppress phosphatidylinositol 3-kinase/Akt signaling via raptor-dependent mTORmediated insulin receptor substrate 1 phosphorylation. Mol. Cell. Biol. 26, 63–76. Vaca, P., Berna, G., Araujo, R., Carneiro, E.M., Bedoya, F.J., Soria, B., and Martin, F. (2008). Nicotinamide induces differentiation of embryonic stem cells into insulin-secreting cells. Exp. Cell Res. 314, 969–974. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K., et al. (2007). A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686. Yin, X., Mead, B.E., Safaee, H., Langer, R., Karp, J.M., and Levy, O. (2016). Engineering stem cell organoids. Cell Stem Cell 18, 25–38. Yuan, H.X., Xiong, Y., and Guan, K.L. (2013). Nutrient sensing, metabolism, and cell growth control. Mol. Cell 49, 379–387.